The invention relates to a method for monitoring metabolic activity of cells in liquid media, to a vessel particularly appropriate for this, and to a respective device for implementing said method.
In the vessels cells to be cultivated as well as a liquid medium are contained wherein the latter concerns with a conventional nutritive solution corresponding to the used cells, if necessary. The solution according to the invention can be employed for the most different cells and the most different studies, in particular in the pharmacological field wherein the metabolic activity of the cells can be monitored over a longer period. For example, monitoring the action with cytotoxic and biocompatibility tests, and optimizing the culture conditions for the production of biological molecules can be carried out.
Commonly, the major nutrient source for cell cultures is glucose which can be converted into lactate by means of aerobic glycolysis or oxidatively with the oxygen consumption and formation of carbon dioxide. Then, many influences of the physiology of a cell have an affect on its metabolic activity such that oxygen consumption is accordingly allowed to vary as well. Starting from the connection between the metabolic activity of cells with respect to the oxygen consumption, and e.g. the glucose consumption and L-glutamine consumption or the generation of lactate it is allowed to conclude the condition of the monitored cells, and as a result the influence of the respective culture conditions.
Based upon these findings, int. al., in xe2x80x9cNoninvasive Oxygen Measurements and Mass Transfer Considerations in Tissue Culture Flasksxe2x80x9d published in Biotechnology and Bioengineering, Vol. 51, pp. 466 to 478, it has been described by Lisa Randers-Eichhorn, how the oxygen consumption of cells cultivated in T-flasks can be determined by means of an optical measurement. Therein, it is suggested to arrange sensor membranes containing fluorescent indicators immediately on the flask bottom, and in the gas space above the nutritive solution within such a T-flask. During the measurement of oxygen concentration with such sensor membranes, the well known physical phenomenon of fluorescent erasure of known fluorescent dyes such as e.g. complexes of Ruthenium (II) is employed due to the influence of oxygen wherein the respective fluorescent intensity changes with the continuous excitation according to the oxygen concentration and the partial pressure of oxygen, respectively. For the determination of oxygen concentration the respective fluorescent intensity immediately, but also the fluorescence life can be measured, and the oxygen concentration can be determined according to a known calibration.
The set-up of measuring instruments described in this document, in particular the arrangement of the sensor membrane on the bottom of the T-flasks, and the neglected determination of some important influence quantities is not suitable to conclude the metabolic activity of the culture cells from oxygen consumption.
The charge of oxygen into the nutritive solution occurs for the most part through the interface of the nutritive solution toward the gas space, therefore here toward the gas space in the T-flask, and the consumption occurs by the cells being present on the bottom of the T-flask. The maximum enabled oxygen concentration which can be achieved within the nutritive solution is the saturation concentration of oxygen CSat (of mg/1) which, according to the equation,
Csat=([pxe2x88x92xcex3*pw(T)]/p0*xcex1(T)*X02
is a function of the total gas pressure p (mbar), the relative humidity of air xcex3 (in values from 0 to 1, wherein 1 corresponds to a value of 100%, and 0 corresponds to a value of 0%), the partial pressure of water vapour pw(T) (mbar) as a function of the temperature T, the mole fraction of oxygen X02 within the gas space of the T-flasks, the Bunsen absorption coefficient xcex1(T) (mg/1) as a function of the temperature T and the normal pressure p0=1013 mbar. Then, it is assumed that the gas space and the nutritive solution have the same temperature, and the gas space has been filled with atmospheric air which chemical composition thereof is sufficiently known. These preconditions are commonly present in breeding chambers in which cells will be cultivated. This saturation concentration of oxygen appears directly below the top surface within the nutritive solution. Therefrom, the oxygen is transported by means of different effects such as diffusion and/or convection toward the cells being present on the bottom. In such a system two quantities are significant. On the one hand, this is the consumption rate kv at which oxygen is consumed by the cells, and on the other hand, the transport rate kT at which oxygen is transported toward the cells. The two quantities are responsible together for that an oxygen gradient results from the top surface of the nutritive solution toward the bottom including cells. If the consumption rate is now slightly smaller than or equal to the transport rate, thus an oxygen concentration comprising a value of 0 is measured with the oxygen membrane on the bottom below the cells. In this case, the cells do not suffer from an oxygen supply since still sufficient oxygen is transported toward the cells, but which does not arrive toward the sensor membrane below the cells, and which, accordingly, cannot be measured any longer. If the consumption rate further increases, e.g. by spreading out the cells, and the consumption rate becomes greater than the transport rate thus this cannot be monitored any longer with the oxygen membrane located on the bottom below the cells. Furthermore, the saturation concentration of oxygen is a very important quantity in addition to the consumption and transport rates as a function of the total gas pressure, the humidity, the temperature and the mole fraction of oxygen and the partial pressure of oxygen, respectively, within the gas space of the T-flask. A change is causing a change of the oxygen gradient, and thus a change of oxygen concentration at any place between the cells and the top surface of the nutritive solution. Since the parameters of total pressure, humidity and temperature have not been determined or checked in the mentioned documentation, it cannot be excluded that measuring results became falsified due to variations of these parameters.
Therefore, it is the object of the invention to predetermine ways wherein monitoring the oxygen consumption and thus the metabolic activity of culture cells can be achieved in a cost effective manner and with an increased accuracy.
According to the invention this object is achieved with the features of claim 1 for a method, and with the features of claim 11 for an appropriate vessel. Advantageous embodiments and improvements of the invention result from the features mentioned in the subordinate claims.
The solution according to the invention is now assuming from that said cells will be cultivated in vessels using a liquid medium wherein as a rule here it concerns with a respective nutritive solution, and that the metabolic activity thereof takes place through the measurement of oxygen concentration at a location within the liquid medium between the cells consuming oxygen and the part being dominant for the oxygen charge into the liquid medium, which is here the top surface of the nutritive solution. Then, the saturation concentration of oxygen in the liquid medium will be determined according to comparison measurements in a vessel of cell cultures without any cells and/or by means of the determination of the parameters of pressure, humidity, temperature and with a chemical composition being well known and constant, of the ambient gas space, which are here the mole fractions of the gas components and the partial pressures thereof, respectively, in the ambient atmosphere. From the comparison between the saturation concentration of oxygen as a set value and the saturation concentration of oxygen at a location of the oxygen gradient within the vessel including the culture cells as an actual value, the oxygen consumption and thus the metabolic activity of the cells are concluded.
For monitoring, vessels can readily be used in contrast to the prior art which comprise an aperture such that the top surface of the liquid medium can be affected by the ambient atmosphere. In certain cases, however, such an aperture is also allowed to be covered and closed, respectively, with a membrane at least being permeable to oxygen such that entering of undesired germs is prevented.
The oxygen concentration will be preferably measured optically with a sensor membrane suitable thereto which optical characteristics thereof change as a function of the respective oxygen concentration. Thus, in a respective vessel at least one suitable sensor membrane should be placed which is located such that it is arranged above the cell cultures cultivated on the vessel bottom, however, below the top surface of the liquid medium.
If the cells cultivated on the vessel bottom are consuming oxygen, the oxygen concentration within the liquid medium will reduce accordingly, and the oxygen concentration actually measured with the sensor membrane will be determined by the oxygen consumption due to the metabolic activity and oxygen quantity which enters into the liquid medium again occurred due to the gradient of oxygen concentration.
Since the conformities to natural laws with respect to the saturation concentration of oxygen in liquid media are relatively properly known, it is possible to calculate the respective saturation concentration of oxygen within a liquid medium under consideration of known parameters which are in particular here the respective temperature, the pressure and the humidity, such that this calculated value of oxygen concentration can be subjected to a value comparison including the actually measured oxygen concentration to evaluate the oxygen consumption and metabolic activity, respectively, of the cell cultures.
However, it is also possible to carry out a reference measurement wherein a second vessel is used in which merely a nutrient medium being completely identical with the used nutritive solutions with respect to the oxygen diffusion characteristics is used. In such a reference vessel a respective sensor membrane is arranged again preferably at the same place whereby the unaffected oxygen concentration can be measured. The reference oxygen concentration thus measured is also allowed to be subjected to a respective value comparison with the oxygen concentration affected by metabolic activity in order to judge the metabolic activity of the culture cells.
Of course, such a value comparison can also be carried out simultaneously for commonly the calculated oxygen concentration and measurement signal of oxygen concentration in the reference vessel including the oxygen concentration measured under metabolic activities moderated by culture cells.
Moreover, the meaningfulness with the benchmarking of metabolic activity of culture cells can be increased when sensor membranes are placed additionally within the vessels in such a manner here again as the oxygen sensitive membranes which include an oxido-reductase on an oxygen sensitive membrane, and wherein the change of oxygen concentration can be measured by a substrate conversion of the enzyme.
Conveniently, these two different sensor membranes should be arranged at least approximately in the same distance of the cell cultures within the liquid medium. For detecting the concentration of the substrate of oxido-reductase and thus the metabolic activity the fluorescent signals of the oxygen membrane and the second oxygen membrane covered with the supplementary membrane are compared. Then, it can be necessary to substract the signal of the first sensor membrane from the signal of the second sensor membrane so as to achieve the sensor response to the enzymatic test of the oxygen, and thus to determine the substrate concentration. Then, it can also be advantageous to introduce a factor by means of which the different oxygen transport relations in the two oxygen membranes are taken into account. Moreover, for an iteration of the signal of the enzymatic sensor it can also be necessary to use different calibration curves depending on the oxygen concentration within the solution, since oxygen is a co-substrate of the enzymatic reaction. In any case, by means of a determination of the enzymatic activity the metabolic activity of the cells can further be determined independent of oxygen consumption of the culture cells.
Since the metabolic activity of the culture cells changes relatively slowly over longer periods, it is sufficient to measure the respective concentrations in longer periods, for example, in intervals of several minutes in order to monitor the metabolic activity of the respective cell cultures with appropriate accuracy, which results in the effort required for a suitable measurement equipment is allowed to be reduced by means of an enabled multiplex operation.
Since the concentration shall be advantageously measured optically it is necessary to use vessels which are optically transparent in definite areas such that the respective change of optical intensity can be measured with optical waveguides (glass fibers), for example, and an appropriate optical sensor. Then, such an optical waveguide has not to be a direct part of a used vessel, or has not to be connected therewith immediately, but can be located and aligned such that it is merely able to detect the area and parts on one sensor membrane with its aperture. As a result, the measuring location and measuring vessel are readily allowed to be separated locally from each other.
Furthermore, there is a possibility as suggested with the way of multiplex measurement to commonly monitor a plurality of vessels in which the same or different cells are cultivated wherein these each can be taken into account individually one after another by means of a respective circuit of at least one multiplexer, respectively. Hence, a more or less spatial resolution type measurement of concentrations can be carried out. For example, sensor membranes can be used which change their absorption and reflection characteristics, respectively, as a function of the oxygen concentration. Alternatively, establishing from known solutions, it is also possible to employ the phenomenon of fluorescence erasure and to use sensor membranes including known fluorescent dyes which are capable to be fluorescent with the light of particular wavelengths when excited wherein the wavelength of the excitation light and the wavelength of the fluorescent light are different.
In the last mentioned case the concentration can be measured once by means of a direct measurement of the respective fluorescent intensity. However, it is more favourable to determine the fluorescence life since in this case aging and subsequently the well known bleaching behaviour do not affect the measuring accuracy.
The oxygen sensitive sensor membranes can be inserted subsequently as well into the vessels useful for the solution according to the invention by means of different techniques. Such sensor membranes can be selectively deposited locally by dispensing, spraying, dipping or glueing as well. Appropriate placing locations within such vessels are, for example, the interior wall of the vessel in a predetermined distance from the vessel bottom, and landing shaped members projecting beyond the bottom surface of the vessel are particularly appropriate wherein on the upper end face thereof a corresponding sensor membrane can be formed and applied, respectively. In this case, at least the landing shaped members are formed from a material being transparent to the relevant wavelengths such that corresponding monitoring can take place from below through the vessel bottom. The remaining vessel parts then have not to be composed conclusively of another transparent material.
Within a vessel to be used in accordance with the invention two of such landing shaped members can be located spaced apart from each other wherein, on the one hand, an exclusively oxygen sensitive sensor membrane is deposited, and on the other hand, such a supplementary membrane can be deposited which is formed with a membrane coated with an enzymatic oxidase sealed against the liquid medium.
For example, if a greater number of samples is to be simultaneously monitored, it is particularly favourable to form a vessel according to the invention in an analogous manner with the known MicroWell(trademark) Plates wherein in such a MicroWell(trademark) Plate a greater number of receiving spaces (cavities) for the cells to be cultivated are located with the liquid medium in a plurality of rows adjacent to each other. A MicroWell(trademark) Plate formed in this manner can be placed in a breeding chamber then, for example, for the cell cultivation, wherein the excitation light and the fluorescent light can be directed from an appropriate light source via optical waveguides upon the sensor membranes, and the fluorescent light from the sensor membranes toward an appropriate optical sensor. Then, it is possible for both the excitation light and the fluorescent light to be guided through a single optical waveguide. Of course, corresponding light guiding for the two different types of light can be carried out inside two separate optical waveguides. For this, the optical waveguides have merely to be fixed and positioned such that their apertures ensure an optimum fluorescence excitation and an approximately complete detection of the fluorescent light. Then, for fixing and positioning the optical waveguides separate mechanism plates can be used which will be dimensioned and aligned relative to the vessels to be used in accordance with the invention, such that the optical waveguides are located and aligned with respect to the sensor membranes. This configuration is particularly appropriate for vessels formed in a MicroWell(trademark) Plate shaped manner. Favourably, a cavity (well) of such a MicroWell(trademark) Plate shaped member can be used for the reference measurement previously mentioned at the beginning, i.e. merely filled with liquid medium but without cell cultures.
The aspect for MicroWell(trademark) Plate shaped members formed and being useful in accordance with the invention or even other appropriate vessels can be formed according to the common laboratory standards such that they are able to be used as well in the conventional form with the different known laboratory instruments.
The spatial resolution type measurement of different samples cannot only take place with the individual optical waveguides associated with the sensor membranes, however, but there is the possibility as well to use an endoscopy array by means of which the image of a greater number of sensor membranes detected by such an array can be directed upon a CCD camera, for example, such that an isochronous spatial resolution type measurement of different oxygen concentrations is enabled.
In the following the invention will be described in more detail by way of example.